Apoptosis, or programmed cell death, is a feature of all multicellular organisms and has critical roles in development, homeostasis, and disease. This intensely studied process is characterized by and requires the activation of a family of proteolytic enzymes known as caspases (cysteine-dependent aspartate-directed proteases). Caspases cleave a large number of specific target proteins, orchestrating the orderly transformation of intact cells into fragments that may be removed from the body by phagocytic cells. Recent evidence points to non-apoptotic roles for caspases in the brain, including the regulation of synaptic plasticity. Caspase activation was recently shown to precede tangle formation in a mouse model of Alzheimer's disease. However, surprisingly little is known about the global scope of neuronal synaptic proteins targeted by caspases, the specific sites of cleavage, and mechanisms by which caspase- mediated cleavages alter neuronal function. This proposal will use an N-terminus labeling approach to positively select cleaved proteins from the complex protein content of synaptosomes or brain lysates, allowing systematic identification caspase targets in synaptosomal preparations as well as in discrete brain regions and models of neuronal apoptotic induction. The analysis will not only identify the protein substrates of caspases in synapses, but will also reveal the precise site of proteolytic cleavage, providing immediate molecular insight into the function of the cleavage event. The information derived from this project will not only stimulate further research on the functional roles of caspase cleavage of synaptic targets, but will also provide immediate candidate biomarkers for human neurological disorders, including trauma, stroke, and neurodegenerative diseases. PUBLIC HEALTH RELEVANCE: Apoptosis, or programmed cell death, as well as the elimination of excess neuronal connections (synapses) is essential for normal brain development. However, the same process may be abnormally re-activated in neurodegenerative disorders such as Alzheimer's Disease. This proposal will use state-of-the-art proteomics methods to globally identify brain synaptic proteins that are cleaved by apoptotic enzymes called caspases. The information derived should provide immediate candidate biomarkers for human neurodegenerative diseases that could help in the diagnosis and monitoring of patients.